1. Field of the Invention
This invention relates to methods for the production of monocotyledonous plants from microspore cultures and from anther cultures. These methods are especially suitable for maize. In other aspects, this invention relates to the regenerated plants, and to seeds and progeny of the regenerated plants derived from cultured anthers or microspores.
2. Description of the Related Art
Ever since the human species emerged from the hunting-gathering phase of its existence, and entered an agricultural phase, a major goal of human ingenuity and invention has been to improve crop yield and to alter and improve the characteristics of plants. In particular, man has sought to alter the characteristics of plants to make them more tasty and/or nutritious, to produce increased crop yield or to render plants more adaptable to specific environments. Recently, plant products which have uses other than as food are becoming increasingly commercially attractive.
Up until recent times, crop and plant improvements depended on selective breeding of plants with desirable characteristics. Initial breeding success was probably accidental, resulting from observation of a plant with desirable characteristics, and use of that plant to propagate the next generation. However, because such plants had within them heterogenous genetic complements, it was unlikely that progeny identical to the parent(s) with the desirable traits would emerge. Nonetheless, advances in controlled breeding have resulted from both increasing knowledge of the mechanisms operative in hereditary transmission, and by empirical observations of results of making various parental plant crosses.
Crop and plant improvement is a major area of commercial interest, for example, monocots L. (corn, maize) is a major worldwide cereal crop. In the continental United States alone, an estimated 70-82 million acres of corn is planted yearly. Unfortunately, current methods for improving inbred corn lines are time consuming, labor intensive, and risky. Seed from those plants shown to retain the desired new characteristics are selected for further breeding. Only after a period of several years can a sufficient seed stock of the inbred corn displaying the desired characteristic be accumulated for commercial breeding purposes. Inbred lines must then typically be crossed to produce hybrids, because inbred lines generally have lower production characteristics, e.g., amount of grain (kernels) per ear, than do hybrids. The process of developing inbred parents generally takes from 4-6 years. Another 3-4 years is required for field testing for a total of 7-10 years.
By use of anther and pollen (microspore) cultures, the time periods to introduce improvements into crops, may be reduced from 4-6 years, to about 1-2 years. Chinese scientists have pioneered this area (Anon., 401 Research Group, 1975). Anthers are the structures that contain the male gametophytes, the haploid microspores or pollen. Microspores have a single nucleus whereas mature pollen have 3 nuclei. These in vitro methods make it possible to test a larger number of new mutations and gene combinations and to select among these for desirable traits. More extensive and imaginative genetic manipulations are also facilitated, such as production of transgenic plants.
Various general methods, including anther and isolated microspore cultures, have been used in a large number of crops, e.g. tobacco and barley, to produce organisms with haploid genetic complements. "Haploids" contain only one-half of the chromosome number present in somatic cells. Somatic cells are those other than gametic cells, the latter being inherently haploid. Haploid complements may also be doubled to produce homozygous diploids. The production of doubled haploid plants is one way to produce homozygous lines. (see review in Kuo, et al. 1985). Haploid plants have both male and female sex organs and produce ears with good seed through self-pollination when chromosomes are doubled. Their seeds may also be used to make F1 crosses to test field performance as hybrids.
As mentioned above, pollen grains develop from microspores which are in turn produced by meiosis in pollen mother cells. The plants resulting from pollen cultures are also usually haploid and may be sterile. To remedy this, the chromosome complement may be doubled by various agents, e.g. colchicine, a mitotic spindle inhibitor, the use of which results in chromosome duplication without cell division. (Jensen, 1974). Occasionally, chromosome doubling occurs spontaneously. The plants resulting from induced or spontaneous chromosome doubling are diploid. Doubling of haploid complements allows homozygous lines to be produced from heterozygous parents in a single generation. Another advantage of this process is that resulting plants breed true, making the selection process for desirable traits more efficient. Microspore culture followed by chromosome doubling is one method of producing doubled haploids.
Factors that affect the frequency of in vitro plant production include genotype, donor plant physiology, the stage of pollen development, pretreatment conditions and the concentrations and nature of ingredients in the media used for culture and regeneration (see review in Keller, et al., 1987). For example, sucrose levels, hormones and additives to culture media affect success. To illustrate the effect of media factors, the effect of one media (N6) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin and other ingredients, is presented by Kuo, supra in his Table 4.
Since the first report by the Chinese of the successful anther culture of maize using an agar solidified medium, improvements have resulted from altering culture conditions, but optimum culture requirements leading to production of plants for commercial use have been elusive. Attempts have been made to adjust media, pretreatment temperature, incubation temperature, and genotype. (Petolino and Jones, 1986). Sorvari et al. (1986) have compared agar-based with starch-based media solidifiers and placed polyester nets on the media to prevent anthers from sinking in. That the response to anther culture is genetically determined was illustrated by the increased response to anther culture of the F.sub.1 progeny plants derived from anther culture, compared to response of the parental lines used for culture. (Petolino et al., 1988).
Instead of culturing entire anthers, isolated microspores may be cultured. Recently, there have been reports of culturing maize isolated microspores (Coumans, et al., 1989; Pescitelli, et al., 1989).
The use of mannitol and of cold temperature pretreatment have been explored separately as a means for increasing the frequency of embryoid formation from isolated microspores. (Wei et al., 1986). Wei et al. applied mannitol pretreatment to isolated barley pollen, but had limited success, possibly because the methods were applied to binucleate pollen, whereas uninucleate pollen (microspores) are believed to be the most responsive in culture. Uninucleate cells most likely lost viability during isolation from the anthers. If mannitol pretreatment was applied to whole anthers containing microspores then results were said to be better.
The ultimate goal of anther and microspore culture is to produce plants. Plant regeneration from in vitro cultures of maize initially used organized tissues for the culture. Plant regeneration from single somatic cells of maize following protoplast isolation was reported by Rhodes et al. (1988), but no seed was recovered. Plant regeneration from isolated microspores has been reported for various cereal species, including corn, (Coumans, et al., 1989; Pescitelli et al., 1989) barley, rice and wheat.
Coumans, et al. pretreated tassels for 14 days at 8.degree. C., and recovered isolated microspores by homogenization of tassel fragments by use of a refrigerated blender. The percentage of microspore-derived plants was low, less than 0.1% of the plated microspores. However, a few plants with viable pollen were produced.
In summary, although some progress has been made in producing plants from anther or isolated microspore culture, "attempts at utilizing haploids in maize breeding have been frustrated by the lack of a reliable means of generating sufficient numbers of doubled haploid lines from a broad spectrum of commercially-useful germplasm." (Petolino et al., 1988). There is a need for improved methods for culturing haploid cells and regenerating fertile plants from the cultured haploids. These methods need to be successful in commercially desirable crop lines.
The inventors have developed new and improved culture techniques for general use in regenerating fertile plants from haploid anther or isolated microspore cultures. They have demonstrated that these methods are successful for commercially desirable lines, for example, those characterized by high production characteristics in maize. The inventors have addressed the major problems in culture of haploids, that is, low initial response frequency as determined by embryo-like growth, difficulties in plant regeneration, and difficulties in chromosome doubling to make diploids from haploids, the latter process leading to fertile plants. The inventors have combined environmental stress conditions which divert the microspores from microsporogenesis to embryogenesis, for example, by combination of stress factors comprising medium components such as mannitol, and cold pretreatments, to recover surprisingly high yields of calli/embryoids and of regenerated fertile plants.